Plants with modified stamen cells

ABSTRACT

A plant, the nuclear genome of which is transformed with a foreign DNA sequence encoding a protein or polypeptide which disrupts the metabolism, functioning and/or development of stamen cells of the plant, said foreign DNA under the control of a stamen-specific promoter. The foreign DNA sequence also optionally encodes a marker.

This application is a continuation of application Ser. No. 07/449,901filed Nov. 22, 1989, now Abandoned, which is a 371 of PCT/EP89/00495filed Apr. 27, 1989, which claims priority to British application8810120.9 filed Apr. 28, 1988.

FIELD OF THE INVENTION

This invention relates to a male-sterile plant and to its reproductionmaterial (e.g., seeds), in which the cells are transformed so that aforeign DNA sequence is stably integrated into their nuclear genome. Theforeign DNA sequence of this invention contains at least one firstforeign DNA (hereinafter the “male-sterility DNA”) that: 1) encodes afirst RNA or protein or polypeptide which, when produced or overproducedin a stamen cell of the plant, disturbs significantly the metabolism,functioning and/or development of the stamen cell; and 2) is in the sametranscriptional unit as, and under the control of, a first promoterwhich is capable of directing expression of the male-sterility DNAselectively in stamen cells of the plant. In particular, this inventionrelates to such a nuclear male-sterile plant and its reproductionmaterial, in which the foreign DNA sequence of this invention is aforeign chimaeric DNA sequence that can also contain at least one secondforeign DNA (the “marker DNA”) that: 1) encodes a second RNA or proteinor polypeptide which, when present at least in a specific tissue orspecific cells of the plant, renders the entire plant easily separablefrom other plants that do not contain the second RNA, protein orpolypeptide at least in the specific tissue or specific cells; 2) is inthe same transcriptional unit as, and under the control of, a secondpromoter which is capable of directing expression of the marker DNA inat least the specific tissue or the specific cells of the plant; and 3)is in the same genetic locus of the nuclear genome of the cells of theplant as the male-sterility DNA.

This invention also relates to a foreign chimaeric DNA sequence thatcontains at least one male-sterility DNA under the control of the firstpromoter and that can also contain, adjacent to the male-sterility DNA,at least one marker DNA under the control of the second promoter.

This invention further relates to a vector that contains the foreign DNAsequence of this invention and is suitable for the transformation ofplant cells, whereby the foreign DNA sequence is stably integrated intothe nuclear genome of the cells.

This invention still further relates to cells of a plant and to plantcell cultures, the nuclear genomes of which are transformed with theforeign DNA sequence.

This invention yet further relates to a process for producing a nuclearmale-sterile plant and its reproduction material and its cell culturescontaining the foreign DNA sequence in which the male-sterility DNA: 1)is under the control of the first promoter and optionally in the samegenetic locus as the marker DNA under the control of the secondpromoter; 2) is stably integrated into the nuclear genome of the plant'scells; and 3) can be expressed selectively in stamen cells of the plantin the form of the first RNA, protein or polypeptide.

The invention further relates to a process for producing hybrid seeds,which grow into hybrid plants, by crossing: 1) the male-sterile plant ofthis invention which includes, in its nuclear genome, the marker DNA,preferably encoding a protein conferring a resistance to a herbicide onthe plant; and 2) a male-fertile plant without the marker DNA in itsgenome. This invention particularly relates to such a process forproducing hybrid seeds on a commercial scale, preferably in asubstantially random population, without the need for extensivehand-labor.

This invention still further relates to a tapetum-specific promoter froma plant genome. This promoter can be used as the first promoter in theforeign DNA sequence of this invention for transforming the plant torender it nuclear male-sterile.

BACKGROUND OF THE INVENTION

Hybridization of plants is recognized as an important process forproducing offspring having a combination of the desirable traits of theparent plants. The resulting hybrid offspring often have the ability tooutperform the parents in different traits, such as in yield,adaptability to environmental changes, and disease resistance. Thisability is called “heterosis” or “hybrid vigor”. As a result,hybridization has been used extensively for improving major crops, suchas corn, sugarbeet and sunflower. For a number of reasons, primarilyrelated to the fact that most plants are capable of undergoing bothself-pollination and cross-pollination, the controlled cross-pollinationof plants without significant self-pollination, to produce a harvest ofhybrid seeds, has been difficult to achieve on a commercial scale.

In nature, the vast majority of crop plants produce male and femalereproductive organs on the same plant, usually in close proximity to oneanother in the same flower. This favors self-pollination. Some plants,however, are exceptions as a result of the particular morphology oftheir reproductive organs which favors cross-pollination. These plantsproduce hybrid offspring with improved vigor and adaptability. One suchmorphology in Cannabis ssp. (hemp) involves male and female reproductionorgans on separate plants. Another such morphology in Zea mays (corn)involves male and female reproductive organs on different parts of thesame plant. Another such morphology in Elaeis quineensis (oilpalm)involves male and fertile female gametes which become fertile atdifferent times in the plant's development.

Some other plant species, such as Ananas comosus (pineapple), favorcross-pollination through the particular physiology of theirreproductive organs. Such plants have developed a so-called“self-incompatibility system” whereby the pollen of one plant is notable to fertilize the female gamete of the same plant or of anotherplant with the same genotype.

Some other plant species favor cross-pollination by naturally displayingthe so-called genomic characteristic of “male sterility”. By thischaracteristic, the plants' anthers degenerate before pollen, producedby the anthers, reach maturity. See: “Male-Sterility in Higher Plants”,M. L. H. Kaul, 1987, in: Monographs on Theoretical and Applied Genetics10, Edit. Springer Verlag. Such a natural male-sterility characteristicis believed to result from a wide range of natural mutations, most ofteninvolving recessive deficiencies, and this characteristic can not easilybe maintained in plant species that predominantly self-pollinate, sinceunder natural conditions, no seeds will be produced.

There are four main types of male sterility observed in nature. All fourtypes of male sterility are used in commercial breeding programs toensure that there is cross-pollination to produce hybrid seed for cropssuch as corn, sugarbeet, oilseed rape and sunflower.

One type of male sterility is nuclear encoded and is believed to beinherited as a recessive allele. For breeding purposes, a recessivemale-sterile parent plant is maintained by crossing it with aheterozygous male-fertile plant that also includes the recessivemale-sterility allele, so that the offspring are 50% recessivemale-sterile plants. The other 50% are male-fertile plants that have tobe rogued out in outcrossing programs which can only be done efficientlyif the recessive male-sterility allele is segregated together with aselectable or screenable marker. In U.S. Pat. No. 4,727,219, a procedureis described for the use of recessive male sterility for the productionof hybrid maize.

A second type of male sterility is nuclear encoded but inherited as adominant allele. An advantage of dominant male sterile plants, ascompared to recessive male sterile plants, is that the dominantmale-sterile plants can be maintained through crossing with amale-fertile plant, to produce offspring that are 50% dominantmale-sterile plants. The usefulness of this dominant nuclearmale-sterile plant is, however, limited because its dominantmale-sterility allele is in most cases not tightly linked (i.e., withinthe same genetic locus) to a selectable or screenable marker.

A third type of male sterility is cytoplasmatically encoded. In mostcases, the cytoplasmic code is in the mitochondrial genome of the plant,and only in a few cases is the code in the chloroplast genome of theplant. The inheritance of cytoplasmatically encoded male sterility doesnot follow Mendelian rules but rather depends on cytoplasmic factors.The offspring obtained from crosses between cytoplasmic male-sterileplants and male-fertile plants all carry the cytoplasmic male-sterilitygene and are therefore sterile. As a result, the offspring of plants ofthis type are only of commercial value if the economic product of theoffspring is not for use as seed but rather for plants such asornamentals and sugarbeet.

A fourth type of male sterility is the result of a combination of bothnuclear encoded male sterility and cytoplasmatically encoded malesterility. The male sterility-inducing nuclear alleles are usuallyrecessive, and only plants that contain the male-sterility cytoplasmicallele and that are homozygous for the male sterility-inducing nuclearallele are phenotypically male sterile. In this type of plant,corresponding dominant male fertility-inducing alleles or “restorers offertility”, produce a male-fertile phenotype. As a result, themale-sterile offspring of this type of plant can be made male-fertile bypollinating the male-sterile plants with pollen containing the restorersof fertility. As a result, the offspring of plants of this type are ofcommercial value where the economic product is seed, that is for plantssuch as corn, sorghum and sunflower.

Typically, hybrid seed production has been accomplished by the largescale planting of cytoplasmic male-sterile plants and male-fertileplants and by somehow (e.g., with a distinctive marker) preventing theresulting hybrid seeds from becoming mixed with non-hybrid seeds.According to U.S. Pat. No. 3,842,538, hybrid seeds are tediouslyseparated from non-hybrid seeds on the basis of color. According to U.S.Pat. No. 4,351,130, the problem of separating hybrid seeds fromnon-hybrid seeds is avoided by using short male-sterile plants and tallmale-fertile plants and then destroying the tall male-fertile plantsafter pollination. According to U.S. Pat. Nos. 4,658,085, 4,517,763 and4,658,084, cytoplasmic male-sterile plants are provided with a herbicidetolerance absent from the male-fertile plants which are destroyed withthe herbicide after pollination. According to U.S. Pat. No. 4,305,225,male-sterile rice plants are sprayed with a growth hormone (e.g.,gibberellin) in order to cause fuller emergence of flower-bearingpanicles from rice leaf sheaths, thereby increasing the ability of theflowers to receive pollen from male-fertile plants.

In all such processes for producing hybrid seeds from male-sterileplants, ways have been sought for simply and. inexpensively obtaining ona commercial scale: 1) high hybrid seed production from eachmale-sterile plant; 2) a hybrid seed population that results almostexclusively from pollen of male-fertile plants and eggs of male-sterileplants and is substantially free of non-hybrid seeds from male-fertileplants; 3) easy production of both the male-sterile and male-fertileplants; and 4) the virtually complete removal or destruction of eitherthe male-fertile plants after they have pollinated the male-sterileplants or the selective separation of non-hybrid seeds, produced by themale-fertile plants, from the hybrid seeds produced by the male-sterileplants.

SUMMARY OF THE INVENTION

In accordance with this invention, a cell of a plant is provided, inwhich the nuclear genome is transformed with a foreign DNA sequence,preferably a foreign chimaeric DNA sequence, characterized by:

(a) a male-sterility DNA encoding a first RNA, protein or polypeptidewhich, when produced or overproduced in a stamen cell of the plant,disturbs; significantly the metabolism, functioning and/or developmentof the stamen cell; and

(b) a first promoter capable of directing expression of themale-sterility DNA selectively in stamen cells of the plant; themale-sterility DNA being in the same transcriptional unit as, and underthe control of, the first promoter.

The foreign DNA sequence in the nuclear genome of the transformed cellcan also comprise, preferably in the same genetic locus as themale-sterility DNA:

(c) a marker DNA encoding a second RNA, protein or polypeptide which,when present at least in a specific tissue or specific cells of theplant, renders the plant easily separable from other plants which do notcontain the second RNA, protein or polypeptide at least in the specifictissue or specific cells; and

(d) a second promoter capable of directing expression of the marker DNAat least in the specific tissue or specific cells; the marker DNA beingin the same transcriptional unit as, and under the control of, thesecond promoter.

Also in accordance with this invention is provided a foreign chimaericDNA sequence that comprises the male-sterility DNA and the firstpromoter and that can also comprise the marker DNA and the secondpromoter, as well as at least one additional DNA encoding a transitpeptide capable of transporting the first protein or polypeptide or thesecond protein or polypeptide into a chloroplast or mitochondria of aplant cell in which the foreign chimaeric DNA sequence is expressed inits cytoplasm.

Further in accordance with this invention are provided: a male-sterileplant and a plant cell culture, each consisting of the transformedcells; a seed of the male-sterile plant; hybrid seeds and plantsproduced by crossing the male-sterile plant with a male-fertile plant;and a process for producing such hybrid seeds.

Still further in accordance with this invention are providedtapetum-specific first promoters.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with this invention, a male-sterile plant is produced froma single cell of a plant by transforming the plant cell in a well knownmanner to stably insert, into the nuclear genome of the cell, theforeign DNA sequence of this invention. The foreign DNA sequencecomprises at least one male-sterility DNA that is under the control of,and fused at its 5′ end to, the first promoter and is fused at its 3′end to suitable transcription regulation signals (including apolyadenylation signal). Thereby, the first RNA, protein or polypeptideis produced or overproduced selectively in stamen cells of the plant soas to render the plant male-sterile. Preferably, the foreign DNAsequence also comprises at least one marker DNA that is under thecontrol of, and is fused at its 5′ end to, the second promoter and isfused at its 3′ end to suitable transcription regulation signals(including a polyadenylation signal). The marker DNA is preferably inthe same genetic locus as the male-sterility, whereby the second RNA,protein or polypeptide is produced in at least the specific tissue orspecific cells of the plant so that the plant can be easilydistinguished and/or separated from other plants that do not contain thesecond RNA, protein or polypeptide in the specific tissue or specificcells. This guarantees, with a high degree of certainty, the jointsegregation of both the male-sterility DNA and the marker DNA intooffspring of the plant.

The cell of a plant (particularly a plant capable of being infected withAgrobacterium) is preferably transformed in accordance with thisinvention, using a vector that is a disarmed Ti-plasmid containing theforeign DNA sequence and carried by Agrobacterium. This transformationcan be carried out using procedures described, for example, in Europeanpatent publications 0,116,718 and 0,270,822. Preferred Ti-plasmidvectors contain the foreign DNA sequence between the border sequences,or at least located to the left of the right border sequence, of theT-DNA of the Ti-plasmid. Of course, other types of vectors can be usedto transform the plant cell, using procedures such as direct genetransfer (as described, for example, in European patent publication0,223,247), pollen mediated transformation (as described, for example,in European patent publication 0,270,356, PCT publication WO85/01856,and European patent publication 0,275,069), in vitro protoplasttransformation (as described, for example, in U.S. Pat. No. 4,684,611),plant RNA virus-mediated transformation (as described, for example, inEuropean patent publication 0,067,553, and U.S. Pat. No. 4,407,956) andliposome-mediated transformation (as described, for example, in U.S.Pat. No. 4,536,475).

Preferably, a nuclear male-sterile plant of this invention is providedby transforming a plant cell with a disarmed Ti-plasmid vectorcontaining the foreign DNA sequence with both a male-sterility DNA underthe control of a first promoter and a marker DNA under the control of asecond promoter. The marker DNA can be upstream or downstream of themale-sterility DNA in the Ti-plasmid vector, but preferably, the two areadjacent to one another and are located between the border sequences orat least located to the left of the right border sequence of theTi-plasmid vector, so that they are properly transferred together intothe nuclear genome of the plant cell. However, if desired, the cell caninitially be transformed with a foreign DNA sequence containing amale-sterility DNA and a first promoter and can subsequently betransformed with a marker DNA and a second promoter, inserted into thesame genetic locus in the cell's nuclear genome as the male-sterilityDNA. Suitable vectors for this purpose are the same as those discussedabove for transforming cells with the foreign DNA sequence. Thepreferred vector is a disarmed Ti-plasmid vector.

The selection of the male-sterility DNA is not critical. A suitablemale-sterility DNA can be selected and isolated in a well-known manner,so that it encodes the first RNA, protein or polypeptide whichsignificantly disturbs the proper metabolism, functioning and/ordevelopment of any stamen cell in which the male-sterility DNA isexpressed, preferably leading thereby to the death of any such stamencell. Preferred examples of male-sterility DNAs encode: RNases such asRNase T1 (which degrades RNA molecules by hydrolyzing the bond after anyguanine residue) and Barnase; DNases such as an endonuclease (e.g.,EcoRI); or proteases such as a papain (e.g., papain zymogen and papainactive protein).

Other examples of male-sterility DNAs encode enzymes which catalyze thesynthesis of phytohormones, such as: isopentenyl transferase which is anenzyme that catalyzes the first step in cytokinin biosynthesis and isencoded by gene 4 of Agrobacterium T-DNA; and the enzymes involved inthe synthesis of auxin and encoded by gene 1 and gene 2 of AgrobacteriumT-DNA. Yet other examples of male-sterility DNAs encode: glucanases;lipases such as phospholipase A₂ (Verheij et al (1981) Rev. Biochem.Pharmacol. 91, 92-203); lipid peroxidases; or plant cell wallinhibitors. Still other examples of male-sterility DNAs encode proteinstoxic to plants cells, such as a bacterial toxin (e.g., the B-fragmentof diphtheria toxin or botulin).

Still another example of a male-sterility DNA is an antisense DNA whichencodes a strand of DNA complementary to a strand of DNA that isnaturally transcribed in the plant's stamen cells under the control ofan endogenous promoter as described, for example, in European patentpublication 0,223,399. Such an antisense DNA can be transcribed into anRNA sequence capable of binding to the coding and/or non-coding portionof an RNA, naturally produced in the stamen cell, so as to inhibit thetranslation of the naturally produced RNA. An example of such anantisense DNA is the antisense DNA of the TA29 gene (described inExample 2) which is naturally expressed, under the control of the TA29promoter, in tapetum cells of the anthers of plants.

A further example of a male-sterility DNA encodes a specific RNA enzyme(i.e., a so-called “ribozyme”), capable of highly specific cleavageagainst a given target sequence, as described by Haseloff and Gerlach(1988) Nature 334, 585-591. Such a ribozyme is, for example, theribozyme targeted against the RNA encoded by the TA29 gene.

Still other examples of male-sterility DNAs encode products which canrender the stamen cells susceptible to specific diseases, such as fungusinfections. Such a male-sterility DNA can be used in a plant wherein allother cells, in which the male-sterility DNA is not expressed, areresistant to the specific disease.

By “foreign” with regard to the foreign DNA sequence of this inventionis meant that the foreign DNA sequence contains a foreign male-sterilityDNA and/or a foreign first promoter. By “foreign” with regard to a DNA,such as a male-sterility DNA and a first promoter, as well a marker DNA,a second promoter and any other DNA in the foreign DNA sequence, ismeant that such a DNA is not in the same genomic environment in a plantcell, transformed with such a DNA in accordance with this invention, asis such a DNA when it is naturally found in the cell of the plant,bacteria, animal, fungus, virus, or the like, from which such a DNAoriginates. This means, for example, that a foreign male-sterility DNAor marker DNA can be: 1) a nuclear DNA in a plant of origin; 2)endogenous to the transformed plant cell (i.e., from a plant of originwith the same genotype as the plant being transformed); and 3) withinthe same transcriptional unit as its own endogenous promotor and 3′ endtranscription regulation signals (from the plant of origin) in theforeign DNA sequence of this invention in the transformed plant cell;but 4) inserted in a different place in the nuclear genome of thetransformed plant cell than it was in the plant of origin so that it isnot surrounded in the transformed plant cell by the genes whichsurrounded it naturally in the plant of origin. A foreign male-sterilityor marker DNA can also, for example, be: 1) a nuclear DNA in a plant oforigin; and 2) endogenous to the transformed plant cell; but 3) in thesame transcriptional unit as a different (i.e., not its own) endogenouspromotor and/or 3′ end transcription regulation signals in a foreignchimaeric DNA sequence of this invention in a transformed plant cell. Aforeign male-sterility or marker DNA can also, for example, be: 1) anuclear DNA in a plant of origin; and 2) endogenous to the transformedplant cell; but 3) in the same transcriptional unit as a heterologouspromotor and/or 3′ end transcription regulation signals in a foreignchimaeric DNA sequence of this invention in a transformed plant cell. Aforeign male-sterility or marker DNA can also, for example, beheterologous to the transformed plant cell and in the sametranscriptional unit as an endogenous promotor and/or 3′ transcriptionregulation signals (e.g., from the nuclear genome of a plant with thesame genotype as the plant being transformed) in a foreign chimaeric DNAsequence of this invention in a transformed plant cell. An example of aforeign male-sterility DNA could come from the nuclear genome of a plantwith the same genotype as the plant being transformed and encode acatalytic enzyme, such as a protease or ribonuclease, that is endogenousto stamen cells of the plant being transformed, so that the enzyme isoverproduced in transformed stamen cells in order to disturbsignificantly their metabolism, functioning and/or development.Preferably, the male-sterility DNA and the marker DNA are eachheterologous to the plant cell being transformed.

By “heterologous” with regard to a DNA, such as a male-sterility DNA, afirst promoter, a marker DNA, a second promoter and any other DNA in theforeign DNA sequence, is meant that such a DNA is not naturally found inthe nuclear genome of cells of a plant with the same genotype as theplant being transformed. Examples of heterologous DNAs includechloroplast and mitochondrial DNAs obtained from a plant with the samegenotype as the plant being transformed, but preferred examples arechloroplast, mitochondrial, and nuclear DNAs from plants having adifferent genotype than the plant being transformed, DNAs from animaland bacterial genomes, and chromosomal and plasmidial DNAs from fungaland viral genomes.

By “chimaeric” with regard to the foreign DNA sequence of this inventionis meant that at least one of its male-sterility DNAs: 1) is notnaturally found under the control of its first promoter for the onemale-sterility DNA; and/or 2) is not naturally found in the same geneticlocus as at least one of its marker DNAs. Examples of foreign chimaericDNA sequences of this invention comprise: a male-sterility DNA ofbacterial origin under the control of a first promoter of plant origin;and a male-sterility DNA of plant origin under the control of a firstpromoter of plant origin and in the same genetic locus as a marker DNAof bacterial origin.

So that the male-sterility DNA is expressed selectively in stamen cellsof a plant, it is preferred that the first promoter, which controls themale-sterility DNA in the foreign DNA sequence, be a promoter capable ofdirecting gene expression selectively in stamen cells of the plant. (By“stamen” is meant the organ of the flower that produces the male gameteand that includes an anther and a filament). Such a stamen-specificpromoter can be an endogenous promoter or an exogenous promoter and canbe from the nuclear genome or from the mitochondrial or chloroplastgenome of a plant cell. In any event, the first promoter is foreign tothe nuclear genome of the plant cell, being transformed. Preferably, thefirst promoter causes the male-sterility DNA to be expressed only inanther, pollen or filament cells, especially in tapetum or antherepidermal cells. The first promoter can be selected and isolated in awell known manner from the species of plant, to be renderedmale-sterile, so that the first promoter directs expression of themale-sterility DNA selectively in stamen cells so as to kill or disablethe stamen and render the plant incapable of producing fertile malegametes. The first promoter is preferably also selected and isolated sothat it is effective to prevent expression of the male-sterility DNA inother parts of the plant that are not involved in the production offertile pollen, especially in female organs of the plant. For example, asuitable endogenous stamen-specific first promoter can be identified andisolated in a plant, to be made male-sterile, by:

1. searching for an mRNA which is only present in the plant during thedevelopment of its stamen, preferably its anthers, pollen or filament;

2. isolating this stamen-specific mRNA;

3. preparing a cDNA from this stamen-specific mRNA;

4. using this cDNA as a probe to identify the regions in the plantgenome which contain DNA coding for the stamen-specific mRNA; and then

5. identifying the portion of the plant genome that is upstream (i.e.,5′) from the DNA coding for the stamen-specific mRNA and that containsthe promoter of this DNA.

Examples of such first promoters are the TA29 promoter, the TA26promoter and the TA13 promoter, hereinafter described in the Examples,which have been isolated from tobacco and are tapetum-specificpromoters. Another tapetum-specific first promoter from another plantspecies can be isolated from its genome, using the TA29, TA26 or TA13gene as a probe as in step 4, above. Under hybridizing conditions, sucha probe will hybridize to DNA coding for a tapetum-specific mRNA in amixture of DNA sequences from the genome of the other plant species(Maniatis et al (1982) Molecular Cloning. A Laboratory Manual. Ed. ColdSpring Harbor Laboratory). Thereafter, as in step 5 above, the othertapetum-specific first promoter can be identified.

If more than one male-sterility DNA is present in the foreign DNAsequence of this invention, all the male-sterility DNAs can be under thecontrol of a single first promoter, but preferably, each male-sterilityDNA is under the control of its own separate first promoter. Where aplurality of male-sterility DNAs are present in the foreign DNAsequence, the male-sterility DNA also can encode the same or differentfirst RNA(s), polypeptide(s) and protein(s). For example, when themale-sterility DNA encodes an RNase such as RNase T1, it preferred thatat least 3, particularly 4 to 6, copies of the male-sterility DNA andits first promoter be provided in the foreign DNA sequence. In anyevent, all the male-sterility DNA(S) and their first promoter(s) arepreferably adjacent to one another in the foreign DNA sequence and inany vector used to transform plant cells with the foreign DNA sequences

The selection of the marker DNA also is not critical. A suitable markerDNA can be selected and isolated in a well known manner, so that itencodes a second RNA, protein or polypeptide that allows plants,expressing the marker DNA, to be easily distinguished and separated fromplants not expressing the second RNA, protein or polypeptide. Examplesof marker DNAs encode proteins that can provide a distinguishable colorto plant cells, such as the A1 gene encodingdihydroquercetin-4-reductase (Meyer et al (1987) Nature 330, 677-678)and the glucoronidase gene (Jefferson et al (1988) Proc. Natl. Acad.Sci. USA (“PNAS”) 83, 8447), or that provide a specific morphologicalcharacteristic to the plant such as dwarf growth or a different shape ofthe leaves. Other examples of marker DNAs confer on plants: stresstolerance, such as is provided by the gene encoding superoxide dismutaseas described in European patent application 88/402222.9; disease or pestresistance such as is provided by a gene encoding a Bacillusthuringiensis endotoxin conferring insect resistance as described inEuropean patent application 86/300291.1 or a gene encoding a bacterialpeptide that confers a bacterial resistance as described in Europeanpatent application 88/401673.4

Preferred marker DNAs encode second proteins or polypeptides inhibitingor neutralizing the action of herbicides such as: the sfr gene and thesfrv gene encoding enzymes conferring resistance to glutamine synthetaseinhibitors such as Biolaphos and phosphinotricine as described inEuropean patent application 87/400,544.0; genes encoding modified targetenzymes for certain herbicides that have a lower affinity for theherbicides than naturally produced endogenous enzymes, such as amodified glutamine synthetase as target for phosphinotricine asdescribed in European patent publication 0,240,792 and a modified5-enolpyruvylshikimate-3 phosphate synthase as a target for glyphosateas described in European patent publication 0,218,571.

The second promoter, which controls the marker DNA, can also be selectedand isolated in a well known manner so that the marker DNA is expressedeither selectively in one or more specific tissues or specific cells orconstitutively in the entire plant, as desired depending on the natureof the second RNA, protein or polypeptide encoded by the marker DNA. Forexample, if the marker DNA encodes an herbicide resistance, it may beuseful to have the marker DNA expressed in all cells of the plant, usinga strong constitutive second promoter such as a 35S promoter (Odell etal (1985) Nature 313, 810-812), a 35S'3 promoter (Hull and Howell (1987)Virology 86, 482-493), the promoter of the nopaline synthetase gene(“PNOS”) of the Ti-plasmid (Herrera-Estrella (1983) Nature 303, 209-213)or the promoter of the octopine synthase gene (“POCS” [De Greve et al(1982) J. Mol. Appl. Genet. 1 (6), 499-511]). If the marker DNA encodesa protein conferring disease resistance, it may be useful to have themarker DNA selectively expressed in wound tissue by using, for example,a TR promoter such as the TR1′ or TR2′ promoter of the Ti-plasmid(Velten et al (1984) EMBO J. 3, 2723-2730). If the marker DNA encodes aherbicide resistance, it may be useful to have the marker DNAselectively expressed in green tissue by using, for example, thepromoter of the gene encoding the small subunit of Rubisco (Europeanpatent application 87/400,544.0). If the marker DNA encodes a pigment,it may be useful to have the marker DNA expressed in specific cells,such as petal cells, leaf cells or seed cells, preferably in the outsidelayer of the seed coat.

One can identify and isolate in a well known manner a tissue-specificsecond promoter for a plant to be rendered male-sterile and easilydistinguishable from non-transformed plants by:

1. searching for an mRNA which is only present in the plant during thedevelopment of a certain tissue, such as its petals, leaves or seeds;

2. isolating this tissue-specific mRNA;

3. preparing a cDNA from this tissue-specific mRNA;

4. using this cDNA as a probe to identify the regions in the plantgenome which contain DNA coding for the tissue-specific mRNA; and then

5. identifying the portion of the plant genome that is upstream from theDNA coding for the tissue-specific mRNA and that contains the promoterfor said DNA.

If more than one marker DNA is present in the foreign DNA sequence ofthis invention, all the marker DNAs can be under the control of a singlesecond promoter, but preferably, each marker DNA is under the control ofits own separate second promoter. More preferably, each marker DNA isunder the control of its own second promoter and encodes a differentsecond RNA, protein or polypeptide, providing different distinguishablecharacteristics to a transformed plant. In any event, the marker DNA(s)and second promoter(s) should be adjacent to each other and to the oneor more male-sterility DNAs contained in the foreign DNA sequence ofthis invention and in any vector used to transform plant cells with theforeign DNA sequence.

It is generally preferred that the first RNA, protein or polypeptide,encoded by the male-sterility DNA, interfere significantly with thestamen cells' metabolism, functioning and/or development by acting inthe cytoplasm or the nucleus of the stamen cells. However, when it isdesired to have the first protein or polypeptide and/or of the secondprotein or polypeptide transported from the cytoplasm into chloroplastsor mitochondria of the cells of transformed plants, the foreign DNAsequence can further include an additional foreign DNA encoding atransit peptide. The additional DNA is between the male-sterility DNAand the first promoter if the first protein or polypeptide is to beso-transported and is between the marker DNA and the second promoter ifthe second protein or polypeptide is to be so-transported. By “transitpeptide” is meant a polypeptide fragment which is normally associatedwith a chloroplast or mitochondrial protein or subunit of the proteinand is produced in a cell as a precursor protein encoded by the nuclearDNA of the cell. The transit peptide is responsible for thetranslocation process of the nuclear-encoded chloroplast ormitochondrial protein or subunit into the chloroplast or themitochondria, and during such a process, the transit peptide isseparated or proteolytically removed from the chloroplast ormitochondrial protein or subunit. One or more of such additional DNA'scan be provided in the foreign DNA sequence of this invention fortransporting one or more first or second proteins or polypeptides asgenerally described in European patent applications 85/402,596.2 and88/402,222.9 and in: Van den Broeck et al (1985) Nature 313, 358-363;Schatz (1987) Eur. J. of Bioch. 165, 1-6; and Boutry et al (1987) Nature328, 340-342. An example of a suitable transit peptide for transportinto chloroplasts is the transit peptide of the small subunit of theenzyme RUBP carboxylase (European patent application 85/402,596.2) andan example of a transit peptide for transport into mitochondria is thetransit peptide of the enzyme Mn-superoxide dismutase (see Example 16).

In the foreign DNA sequence of this invention, 3′ transcriptionregulation signals can be selected among those which are capable ofenabling correct transcription termination and polyadenylation of mRNAin plant cells. The transcription regulation signals can be the naturalones of the gene to be transcribed but can also be foreign orheterologous. Examples of heterologous transcription regulation signalsare those of the octopine synthase gene (Gielen et al (1984) EMBO J. 3,835-845) and the T-DNA gene 7 (Velten and Schell (1985) Nucleic AcidsResearch (“NAR”) 13, 6981-6998).

Also in accordance with this invention, plant cell cultures, such asanther cell cultures, containing the foreign DNA sequence of thisinvention in which the first promoter effects expression of themale-sterility DNA at a given stage of pollen development, moreespecially after meiosis, can be used to regenerate homozygous dominantmale-sterile plants (“Efficient isolation of microspores and theproduction of microspore-derived embryos from Brassica napus”, E. B.Swanson, M. P. Coumans, S. C. Wu, T. L. Barby and W. D. Beversdorf,Plant Cell Reports (1987) 6: 94-97).

Further in accordance with this invention, processes are provided forproducing hybrid seeds which can be grown into hybrid plants. Oneprocess involves crossing a nuclear male-sterile plant including atleast one marker DNA with a male-fertile plant without the marker DNA.Both male-sterile and male-fertile plants are planted in separate rowsnear to each other. Another process involves crossing a nuclearmale-sterile plant including at least two different marker DNAs with amale-fertile plant including, in common, only one of the two differentmarker DNAs in a homozygous form. Both male-sterile and male-fertileparent plants can be grown in a substantially random population,increasing the chances of cross-pollination, without the need forprecise planting patterns. The male-fertile parent plant can thereafterbe easily removed from the population, using the distinctive traitencoded by the non-common marker DNA which is not possessed by themale-fertile parent plant. Preferably in this process, the non-commonmarker DNA in the male-sterile plant is under the control of aconstitutive promoter and encodes a protein or polypeptide that rendersthe male-sterile plant resistant to a particular herbicide. Themale-fertile plant can then be destroyed after cross-pollination, usingthe particular herbicide.

Plants, transformed with the male-sterility DNA, preferably with boththe male-sterility DNA and the marker DNA encoding herbicide-resistance,stably integrated and transmissible throughout generations as dominantalleles in accordance with this invention, are alternatives to, andprovide several advantages over, presently used cytoplasmicmale-sterility systems for breeding and producing hybrid crops. Suchadvantages include:

1. For cross-pollinating crops, the breeding strategy is muchsimplified, because it is not necessary to introduce a restorer geneinto the male-fertile parent line of the cross that will produce thecommercially sold hybrid seed. Indeed, a heterozygous nuclearmale-sterile parent line crossed with another male-fertile parent linefor commercial seed production will produce 50% male-sterile hybridoffspring and 50% male-fertile hybrid offspring, as a result of whichthe commercial crop will produce enough pollen to guarantee full seedset and therefore normal yield. Examples for such crops are corn andoilseed rape.

2. For crops for which the seeds do not represent the economic harvest,the breeding strategy is also much simplified without the need of arestorer gene expressed in the male-fertile parent line. Indeed, forthese crops it does not matter that 50% of the commercially sold hybridseeds are male-sterile. Examples for these crops are sugarbeet andalfalfa.

3. The system allows production of nuclear male-sterile lines andmaintainer lines from existing inbred lines in one operation,eliminating the need for backcrossing. This reduces the time lag betweenconception and commercialization of a hybrid by at least 6 to 8generations. An example of a typical strategy for producing hybridplants using as parent plant the plants having inserted and expressingthe male-sterility DNA may consist of the following steps:

1) making test hybrids by hand, by crossing inbred lines, and testingfor combining ability and selected characteristics (2 years).

2) making one parent line of each of the selected hybrids nuclearmale-sterile by the process which is the object of this invention (1year).

3) multiplying the nuclear male sterile parent plant obtained from saidprocess, hereinafter called “A^(S)”, and its maintainer line,hereinafter called “A”, and the pollinating male-fertile parent plant,hereinafter called “B”, of the future commercial crop (3 years). Duringthe same period, introducing the selected hybrids in official yieldtrials (3 years).

4) producing and selling the approved hybrid seed (1 year).

4. Combined with a marker DNA encoding herbicide-resistance, such anuclear male-sterility system allows production of 2-, 3- and 4-wayhybrids in any combination required. It is believed to be sufficient tointroduce the male-sterility DNA and adjacent thereto the marker DNAinto the nuclear genome of one plant which will be used as one of thegrandparent breeding lines for obtaining 2- or 3-way hybrids, and intothe nuclear genome of two plants which will be used as the twograndparent lines for 4-way hybrids. Each breeding line can bemaintained by the following two crosses given here by way of example,and whereby “SH” stands for the dominant alleles respectively ofmale-sterility (S) and herbicide resistance (H), and sh stands for therecessive alleles respectively of male fertility (s) and herbicidesensitivity (h):

a. SH/sh×sh/sh gives 50% SH and 50% sh offspring, and after sprayingwith the herbicide to which H confers resistance, 100% sterile seedlingsare obtained.

b. sh/sh×sh/sh gives 100% fertile offspring.

5. It provides a protection for the owner of the marker DNA that hasbeen integrated into the male-sterility system by making it moredifficult for competitors to breed the marker DNA into their ownbreeding lines.

For illustrative purposes, two crop breeding schemes in accordance withthis invention are given as follows:

Scheme 1: Breeding a Plant Containing Adjacent Male-sterility DNA andMarker DNA Encoding Herbicide-resistance

1A) maintaining the male-sterility line A^(S):

line A^(SH/sh)×line A^(sh/sh)

giving

50% A^(SH/sh) (phenotype: male-sterile, herbicide-resistant)

50% A^(sh/sh) (phenotype: male-fertile, herbicide-susceptible)

1B) producing the hybrid seed crop:

a) planting seeds of B^(sh/sh) (male plants) and the seeds obtained bythe cross 1A) consisting of A^(SH/sh) and A^(sh/sh) (“female” plants) inseparate rows.

b) eliminating the genotype A^(sh/sh) by spraying the female rows withthe herbicide.

c) cross-pollination occurring:

A^(SH/sh)×B^(sh/sh) and B^(sh/sh)×B^(sh/sh)

giving in the female rows:

50% AB^(SH/sh) (phenotype: hybrid, male-sterile, herbicide-resistant)

50% AB^(sh/sh) (phenotype: hybrid, male-fertile, herbicide-sensitive)

 and in the male rows: 100% B^(sh/sh).

d) eliminating the genotype B^(sh/sh) occurring in the male rows byspraying with the herbicide or by mechanical means.

e) harvesting the hybrid seeds of the female rows wherein thecross-pollination of c) occurred. This is the commercially sold seed.

Scheme 2: Breeding a Plant Containing Adjacent Male-sterility DNA andTwo Marker DNAs, each Encoding a Different Herbicide-resistance (H1 andH2).

2A) maintaining the male-sterile line A^(S):

A^(S):A^(SH1H2/sh1h2)×A^(sh1h2/sh1h2)

giving

50% A^(SH1H2/sh1h2) (phenotype: male-sterile, resistant to bothherbicides).

50% A^(sh1h2/sh1h2) (phenotype: male-fertile, susceptible to bothherbicides).

2B) maintaining pollination line B:

B^(sh1H2/sh1H2)×B^(sh1H2/sh1H2)

giving

100% B^(sh1H2/sh1H2) (phenotype: male-fertile, susceptible to herbicide1 and resistant to herbicide 2).

2C) producing the hybrid seed crop:

a) planting the seeds obtained from 2A) and the seeds obtained from 2B)at random.

b) eliminating the genotype A^(sh1h2/sh1h2) by spraying the field withherbicide 2.

c) cross-pollination occurring:

A^(SH1H2/sh1h2)×B^(sh1H2/sh1H2)

giving

50% AB^(SH1H2/sh1H2)

50% AB^(sh1h2/sh1H2)

 and

self-pollination occurring:

B^(sh1H2/sh1H2)×B^(sh1H2/sh1H2)

giving

100% B^(sh1H2/sh1H2)

d) eliminating plants with genotype B^(sh1H2/sh1H2) obtained from theparent line B, for which self-pollination occurred, by spraying thefield with herbicide 1.

e) harvesting hybrid seeds of the remaining plants A^(SH1H2/sh1H2)obtained by the cross-pollination of c).

The following Examples illustrate the invention. The figures referred toin the Examples are as follows:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows restriction maps of TA29 cDNA and its ClaI fragment inpTA29S3 of Example 1.

FIG. 2 shows the cDNA sequence of the PstI fragment of the TA29 gene ofExample 2.

FIG. 3A shows the DNA sequence and amino acid sequence of the TA29 gene,from its ClaI site to its Hind III site. Above the sequences, theimportant restriction sites are indicated, and under the sequences isthe amino acid sequence encoded by the ORF. Also indicated are:

from nucleotide (“nt”) 1446 to 1452: TATA box (asterisks),

at nt 1477: transcription initiation site of TA29 mRNA (asterisk),

from nt 1514 to 1537: the 3′ to 5′ sequence of a synthetic oligomer asdescribed in Example 2, and

from nt 1940 to 2296 (between arrows);

the aligned sequence of TA29 cDNA.

FIG. 3B shows the alignment of the TA13 cDNA (top line) and the TA29cDNA (bottom line); as discussed in Example 4. Homologous nucleotidesare indicated by vertical lines.

FIG. 3C shows the sequence of the TA26 cDNA, as discussed in Example 4;the ORF is underlined.

FIG. 4A shows schematically the construction of the vector pMB2 ofExample 3.

FIG. 4B shows a map of the vector pMB3 of Example 3.

FIG. 5 shows a map of the vector pTTM3 of Example 5.

FIG. 6 shows a map of the vector pTTM4 of Example 7.

FIG. 7A shows a map of the vector pTTM6 of Example 9.

FIG. 7B shows a map of the vector PTTM6A⁻ of Example 11.

FIG. 8 shows a map of the vector pTTM8 of Example 12.

FIG. 9A shows a map of the vector pTVEP1 of Example 14.

FIG. 9B shows a map of the vector pTVEP2 of Example 14.

FIG. 10A shows a map of the vector pTVEP63 of Example 16.

FIG. 10B shows a map of the vector pTVEP62 of Example 16.

FIG. 11 shows a photograph of flowers of normal tobacco plants comparedwith flowers of tobacco plants transformed with the male-sterility DNAof Example 9.

FIG. 12 shows a photograph of a transverse cutting of the anther of anormal tobacco plant compared with the anther of a tobacco planttransformed with the male-sterility DNA of Example 9 (enhancement:×250).

Unless otherwise stated in the Examples, all procedures for making andmanipulating recombinant DNA were carried out by the standardizedprocedures described in Maniatis et al, Molecular Cloning—A LaboratoryManual, Cold Spring Harbor Laboratory (1982). The following plasmids andvectors, used in the Examples, have been deposited in the DeutscheSammlung Für Mikroorganismen und Zellculturen (“DSM”), Mascheroder Weg1B, D-3300 Braunschweig, Federal Republic of Germany under theprovisions of the Budapest Treaty:

Plasmid or DSM Acession vector No. Date pMB3 4470 21 Mar. 1988 pGSC16004467 21 Mar. 1988 pGSC1700 4469 21 Mar. 1988 pGV2260 2799 Dec. 1983pGSC1701A 4286 22 Oct. 1987 pTTM4 4471 21 Mar. 1988 pTTM6 4468 21 Mar.1988

DETAILED DESCRIPTION OF THE INVENTION EXAMPLE 1

Subcloning of an Anther-specific Gene (the “TA29 Gene”)

From Professor Robert Goldberg of the University of California, LosAngeles (UCLA) were obtained: a Nicotiana tabacum anther-specific cDNA(“TA29 cDNA”) cloned as a PstI fragment in pBR329 (Covarrubias andBolivar (1982) Gene 17, 79) by GC tailing; and the corresponding genomicclone (“lambda TA29”) that was isolated from a N. tabacum “Samsun”genomic library using TA29 cDNA as a probe and that was inserted in theEcoRI site of the lambda phage vector cH32 (Loenen and Blattner (1983)Gene 26, 171). The TA29 cDNA was 365 base pairs long (±0.4 kb) andhybridized to a tapetum-specific mRNA of 1,100 nucleotides whichaccounts for 0.24% of the poly A⁺ mRNA from anthers of the N. tabacum.As shown in FIG. 1, lambda TA29 contains two EcoRI fragments, the totalinsert measuring 13.2 kb.

An internal 7.5 kb ClaI fragment as shown in FIG. 1, containing the TA29gene, was subcloned from lambda TA29 in pLK31 (Botterman and Zabeau(1987) DNA 6, 6) which produced a plasmid named “pTA29S3”.Nitrocellulose bound fragments of lambda TA29, digested with thecombination of EcoRI/ClaI/HindIII/HindIII-EcoRI and the combination ofClaI-EcoRI and hybridized against TA29 cDNA, indicated the presence ofsequences homologous to TA29 cDNA.

EXAMPLE 2

Nucleotide Sequence Determination of TA29 cDNA and its HomologousSequence from pTA29S3; Mapping of TA29 Gene and its Promoter

The PstI insert of TA29 cDNA in pBR329 was completely sequenced (Maxamand Gilbert (1977) Proc. Natl. Acad. Sci. USA (“PNAS”) 74, 560). ThecDNA sequence is shown in FIG. 2. It reveals the presence of one openreading frame over the entire cDNA sequence (as indicated).

Then, the sequence of the ClaI insert in pTA29S3 was determined from theClaI site to the HindIII site (3261 base pairs apart). Comparison of theTA29 cDNA sequence and the pTA29S3 sequence revealed the presence of asequence in the pTA29S3 which was completely homologous with the TA29cDNA sequence.

FIG. 3 shows the sequence of the TA29 gene in pTA29S3. The sequence inpTA29S3 that is identical to the TA29 cDNA sequence is between thearrows in FIG. 3. A putative open reading frame is revealed by thecorresponding amino acid sequence in FIG. 3. This indicates that theTA29 gene encodes a protein of 321 amino acid residues and that thereare no introns present in the coding region. The length of the openreading frame of 964 (+leader) nucleotides matches the size of atranscript present in tobacco anther mRNA prepared from anthers isolatedfrom young (12-20 mm long) tobacco flower buds and absent in the mRNAisolated from leaf and older flowers (when the buds are opened andpetals have appeared). The size of this mRNA is approximately 1100nucleotides.

There are two ATG codons, one at nucleotide (“nt”) 1527 and the other atnt 1560, which could serve as initiation codon for the open readingframe, 33 nucleotides apart. There is a consensus sequence TATA at nt1446 present 81 nucleotides 5′ upstream of the first ATG codon(indicated by asterisks in FIG. 3). To confirm that this “TATA” box ispart of the promoter of the TA29 gene, the 5′ end of the TA29 mRNA wasdetermined. This was done by primer extension (Mc Knight et al (1981)Cell 25, 385). For this purpose, an oligomer of 24 nucleotides, havingthe sequence: 5′ GGA GCT ACC ATT TTA GCT AAT TTC 3′, was used as it iscomplementary to the TA29 gene from nt 1514 to nt 1537 as shown in FIG.3.

This oligonucleotide was ³²P labeled by kination at the 5′ end. Afterbeing hybridized with anther mRNA, the oligonucleotide was extended byreverse transcriptase. The resulting extended oligonucleotide wasanalyzed on a sequencing gel, next to a sequencing ladder, to determineits exact size. The fragment was shown to be 61 nucleotides long. Thisindicates that transcription initiation of the TA29 mRNA occurred at nt1477 (indicated by asterisk in FIG. 3). Hence, the TA29 gene has a TATAbox located 31 nucleotides upstream of the transcription initiationsite. The mRNA contains a 51 nucleotide-long leader sequence from nt1477 to nt 1527, a coding region of 964 nucleotides from nt 1527 to nt2491, and a 3′ non coding region of approximately 100 nucleotides fromnt 2492 to nt 2590. As is the case in approximately 92% of presentlycharacterized plant genes (Joshin (1987) Nucleic Acids Research (“NAR”)15 (16), 6643), it is believed that the first AUG codon of the mRNA isused to initiate translation. The TA29 promoter thus appears to belocated between the ClaI restriction site and nt 1477.

EXAMPLE 3

Construction of a Promoter Cassette (“PTA29”) derived from the TA29 Gene

To construct chimaeric DNA sequences containing the 5′ regulatorysequences, including the promoter, of the TA29 gene in the sametranscriptional unit as, and controlling, a first heterologousmale-sterility DNA, a cassette was constructed as shown in FIG. 4 bysubcloning a 2.5 kb ClaI/AccI fragment from pTA29S3 into the polylinkerAccI site of the pMAC 5-8 vector system (European patent application87/402348.4). This produced a vector named “pMB2”, shown in FIG. 4,which could be used to isolate single strand DNA for use in sitedirected mutagenesis.

Then, the sequence surrounding the first ATG codon AAAATGGTA wasmodified to ACCATGGTA by substituting two adenine residues for cytosineresidues. This mutation created the sequence CCATGG which is therecognition site for the restriction enzyme NcoI. This site directedmutagenesis in pMB2 was performed using a synthetic oligonucleotide of24 nucleotides with the following sequence:

3′GTT TAA TCG ATG GTA CCA TCG AGG 5′

The resulting plasmid, containing the newly created NcoI site, was named“pMB3” and is shown in FIG. 4 bis. The precise nucleotide sequencespanning the NcoI site was determined in order to confirm that it onlydiffered from the 5′ sequence of the TA29 gene by the AA—CCsubstitution, creating the NcoI site. The 1507 nucleotide long fragmentClaI—NcoI was named “PTA29”.

EXAMPLE 4

Identification of cDNA Clones Obtained from Other Stamen-specific mRNAs

To demonstrate that other anther-specific mRNAs could be identified andthen used to isolate cDNA clones with analogous properties to the TA29gene, two other N. tabacum anther-specific cDNAs (“TA13 cDNA” and “TA26cDNA”) were obtained from Professor Goldberg of UCLA.

TA13 cDNA is a clone of 1100 bp which hybridized to two mRNA species ofabout 1100 and 1200 nucleotides, respectively, which are specific fortapetum cells and are abundant at a very early stage of antherdevelopment. TA13 cDNA was sequenced, using the procedure of Example 2,and then compared with the sequence of TA29 cDNA as shown in FIG. 3B.This sequence comparison reveals that TA13 cDNA and TA29 cDNA share 92%homology, and the ORF is very rich in glycine content.

TA26 cDNA was cloned as a PstI insert into pBR329 by poly-G/C tailing.It is a clone of 519 bp which hybridized to one tobacco mRNA species of580 nucleotides, which mRNA is specific for tapetum cells and abundantat a certain stage of anther development. The entire TA26 cDNA wassequenced, using the procedure of Example 2, and when compared with thesequence of TA29 cDNA, revealed no homology. The sequence of TA26 cDNAis given in FIG. 3C.

EXAMPLE 5

Construction of a Chimaeric DNA Sequence of PTA29 and a GlucuronidaseGene

A plasmid named “pTTM3”, shown in FIG. 5, was constructed by assemblingthe following well known DNA fragments with PTA29:

1. a vector fragment, including T-DNA border sequences, derived frompGSC1600;

2. a chimaeric sequence containing the promoter cassette PTA29 fromExample 3, fused in frame with a pMB3 NcoI/EcoRI fragment containing anE. coli gene encoding beta-glucuronidase (“GUS” [Jefferson et al (1986)PNAS 83, 8447; Jefferson et al (1987) EMBO J. 6, 3901]) and the 3′ endsignals of an octopine-synthase gene (“OCS” [Dhaese et al (1983) EMBO J.2, 419]);

3. a chimaeric sequence containing an Arabidopsis SSU promotor (“PSSU”or “PSSUARA”), a herbicide resistance gene sfr (European patentapplication 87/400,544.0) and the 3′ end signals of a T-DNA gene 7(Velten and Schell (1985) NAR 13, 6981); and

4. a chimaeric sequence containing the EcoRI/SacI fragment from pGSFR401which contains a nopaline-synthase promoter (“PNOS”), a neo geneencoding kanamycin resistance and the 3′ end signals of an octopinesynthase gene (European patent application 87/400,544.0, whereinpGSFR401 is called “pGSR4”).

pTTM3 is a T-DNA vector containing, within the T-DNA border sequences,two chimaeric sequences: PSSU-sfr in which the sfr is a marker DNA(European patent application 87/400,544.0) under the control of PSSU asa second promoter; and PTA29-GUS in which GUS is a reporter gene whoseexpression in plants and plant cells under the control of the TA29promoter can easily be localized and quantified.

EXAMPLE 6

Introduction of the Chimaeric DNA Sequence of Example 5 into Tobacco

A recombinant Agrobacterium strain was constructed by mobilizing pTTM3(from Example 5) from E. coli into Agrobacterium C58C1 Rif^(R)containing pGV2260 (De Blaere et al (1985) NAR 13, 4777). Mobilizationwas carried out using E. coli HB101 containing pRK2013 (Figurski et al(1979) PNAS 76, 1648) as a helper as described in European patentpublication 0,116,718. The resulting Agrobacterium strain contained ahybrid Ti-plasmid comprising pGV2260 and pTTM3.

This strain was used to transform tobacco leaf discs (N. tabacum PetiteHavane SR1) using standard procedures as described, for example, inEuropean patent application 87/400,544.0. Transformed calli and shootswere selected using 5 mg/l of the herbicide phosphinothricin in themedium (De Block et al (1987) EMBO J. 6, 2513). No beta-glucuronidaseenzyme activity was detected in the transformed herbicide-resistantcalli and shoots.

Then, the transformed shoots were rooted, transferred to soil in thegreenhouse and grown until they flowered. The flowers were examined, andonly the tapetum cells in the anthers of the stamen were found tocontain beta-glucuronidase activity. This shows that the TA29 promoteris capable of directing expression of a heterologous gene, like thebeta-glucuronidase gene, selectively in tapetum cells of the plants.

EXAMPLE 7

Construction of a Chimaeric DNA Sequence of PTA29 and a Gene 4

A plasmid named “pTTM4”, shown in FIG. 6, was constructed by assemblingthe following well known DNA fragments with PTA29:

1. a vector fragment, including T-DNA border sequences, derived frompGSC1700 (Cornellisen and Vandewiele (1989) NAR 17 (1), 19-29);

2. the chimaeric sequence (no. 3) of Example 5, containing the PSSUpromotor controlling expression of herbicide-resistance gene sfr and the3′ end of a T-DNA gene 7;

3. the chimaeric sequence (no. 4) of Example 5, containing the PNOSpromoter controlling expression of the neo gene and the 3′ end of theoctopine synthase gene; and

4. a chimaeric sequence containing the PTA29 promotor cassette fromExaimple 3, fused in frame with an Agrobacterium T-DNA gene 4 thatencodes isopentenyl transferase (Akiyoshi et al (1984) PNAS 76, 5994;Barry et al (1984) PNAS 81, 4776) containing its own 3′ endtranscription regulation signals.

pTTM4 is a binary type T-DNA vector containing, within the T-DNA bordersequences, the following chimaeric sequences: PSSU-sfr and PNOS-neo inwhich the sfr and neo genes are marker DNAs that encode dominantselectable markers for plants and that are under the control ofrespectively PSSU and PNOS as second promoters; and PTA29-gene 4 inwhich gene 4 is a male-sterility DNA that is under the control of PTA29as a first promoter and encodes the enzyme isopentenyl transferase whichwill cause the enhanced production of cytokinin. Enhanced cytokininproduction in tapetum cells, under the control of the TA29 promoter,will disturb the metabolism and organogenesis of the tapetum cells.

EXAMPLE 8

Introduction of the Chimaeric DNA Sequence of Example 7 into Tobacco

As described in Example 6, pTMM4 (from Example 7) was introduced withmobilization from E. coli into Agrobacterium C58C1 Rif^(R). Theresulting Agrobacterium strain contained a binary type Ti-plassidcomprising pGV2260 and pTTM4.

As also described in Example 6, this strain was used to transformtobacco leaf discs, and transformed calli and shoots were selected using5 mg/l of phosphinothricin. Transformed herbicide-resistant shoots wererooted, which shows that gene 4 was not yet being expressed in thetransformed plants.

The plants were then transferred to soil in the greenhouse and grownuntil they flower. The flowers are examined, and no functional tapetumcells are found in their anthers of their stamen. This shows that theTA29 promoter is capable of directing expression of the heterologousgene 4 selectively in tapetum cells of the plants.

EXAMPLE 9

Construction of a Chimaeric DNA Sequence of PTA29 and a RNAse T1 Gene

A plasmid named “pTTM6”, shown in FIG. 7A, was constructed by assemblingthe following well known DNA fragments with PTA29:

1. a vector fragment, including T-DNA border sequences, from pGSC1600;

2. the chimaeric sequence (no. 3) of Example 5, containing the PSSUpromotor, the herbicide resistance gene sfr and the 3′ end of the T-DNAgene 7; and

3. a chimaeric sequence, containing the pTA29 promoter cassette fromExample 3, fused in frame with a synthetic gene encoding RNase T1 fromA. orhyzae, (Quaas et al, “Biophosphates and their Analogues-Synthese,Structure, Metabolism and Activity” (1987) Elsevier Science Publisher B.V., Amsterdam; Quaas et al (1988) Eur. J. Biochem. 173, 617-622.) andthe 3′ end signals of a nopaline synthase (“NOS”) gene (An et al (1985)EMBO J. 4 (2), 277).

pTTM6 is a T-DNA vector containing, within the T-DRA border sequences,two chimaeric sequences; PSSU-sfr which is a marker DNA under thecontrol of PSSU as a second promoter; and PTA29-RNase T1 gene which is amale-sterility DNA under the control of PTA29 as a first promoter.Expression in tapetum cells of the male-sterility DNA under the controlof the TA29 promoter will produce RNase T1 that will be lethal for thecells, since the RNase T1 will degrade the RNA molecules which areindispensable for these cells'metabolism.

EXAMPLE 10

Introduction of the Chimaeric DNA Sequence of Example 9 into Tobacco

As described in Example 6, a recombinant Agrobacterium strain wasconstructed by mobilization of pTTM6 (from Example 9) from E. coli intoAgrobacterium C58C1 Rif^(R). The resulting Agrobacterium strain,harboring a cointegrated Ti-plasmid comprised of pGV2260 and pTTM6, wasused for transforming tobacco leaf discs. Transformed calli and shootswere selected using 5 mg/l phosphinothricin. That the RNase T1 gene wasnot expressed in the transformed herbicide-resistant calli and shootswas shown by their growth.

The transformed shoots were rooted, transferred to soil in thegreenhouse and grown until they flowered. The transformed tobacco plantsdeveloped normal flowers except for their anthers. The anthers, althoughof normal shape, dehisched later in time, compared to the anthers ofnon-transformed tobacco plants (see FIG. 11). Upon dehiscense, eitherlittle or no pollen was released from the transformed plants, and thepollen grains formed by the transformed plants, were about 50 to 100times smaller in volume than normal pollen grains and were irregularlyshaped. Moreover, most of the pollen grains from transformed plantsfailed to germinate, and the germination efficiency of pollen fromtransformed plants was about 0 to 2% of the germination efficiency ofnormal pollen grains. Furthermore, the transformed plants did notproduce any seeds by self-pollination—neither by naturalself-pollination nor by hand-provoked self-pollination.

Microscopic evaluation, by thin layer cross section, of a transformedplant showed that no normal tapetum layer was formed and that the pollensack remained empty (see FIG. 12). This shows that the TA29 promoter iscapable of directing expression of the heterologous RNase T1 geneselectively in tapetum cells of the transformed plants, and that theRNase T1 is capable of sufficiently disturbing the functioning of thetapetum cells, so as to render the plants male-sterile.

EXAMPLE 11

Introduction of a Derivative of the Chimaeric DNA Sequence of Example 9into Oilseed Rape

A recombinant Agrobacterium strain was constructed by mobilization ofpTTM6A⁻ from E. coli into Agrobacterium C58 Rif^(R) containing pMP90(Koncz and Schell (1986) Mol. Gen. Genetics 204, 383-396). pMP90provides vir and trans functions and does not carry a gene encodingampicillin resistance. As shown in FIG. 7B, pTTM6A⁻ is a derivative ofPTTM6 (from Example 9), in which the β-lactamase gene encodingampicillin resistance has been inactivated by insertion of a DNAsequence into the ScaI site of the β-lactamase gene.

The resulting Agrobacterium strain (named “A3144”), harboring pMP90 andpTTM6A⁻, was used for the transformation of Brassica napus according tothe procedure of Lloyd et al (1986) Science 234, 464-466 andKlimaszewska et al (1985) Plant Cell Tissue organ Culture 4, 183-197.Carbenicillin was used to kill A3144 after co-cultivation occurred.Transformed calli were selected on 5 mg/l phosphinotricine and 100 ug/mlkanamycin, and resistant calli were regenerated into plants. Afterinduction of shoots and roots, the transformants were transferred to thegreenhouse and grown until they flower. The flowers are examined, andthey exhibit essentially the same phenotype as was observed for thetransformed tobacco plants described in Example 10. This shows that theTA29 promoter is capable of directing the expression of the heterologousRNase T1 gene selectively in tapetum cells of plants other than tobacco,so as to render such other plants male-sterile.

EXAMPLE 12

Construction of a Chimaeric DNA Sequence of PTA29 and a Barnase Gene

A plasmid named “pTTM8” shown in FIG. 8, was constructed by assemblingthe following well known fragments with PTA29:

1. a vector fragment, including T-DNA border sequences derived frompGSC1700 (Cornelissen and Vandewiele (1989) NAR 17 (1) 19-29) and inwhich the β-lactamase gene (1′ of FIG. 8) has been inactivated byinsertion of a DNA sequence into its ScaI site;

2. the chimaeric sequence (no. 3) of Example 5, containing the PSSUpromoter, the herbicide-resistance gene sfr and the 3′ end of T-DNA gene7;

3. the chimaeric sequence (no. 4) of Example 5, containing the PNOSpromoter, the neo gene, and the 3′ end of the octopine synthase gene;and

4. a chimaeric sequence, containing the PTA29 promoter cassette fromExample 3, fused in frame with the Barnase gene from Bacillusamiloliquefaciens (Hartley and Rogerson (1972) Preparative Biochemistry2, (3), 243-250) and the 3′ end of the nopaline synthase gene of Example9.

pTTM8 is a binary type T-DNA vector containing, within the T-DNA bordersequences, three chimaeric sequences: PSSU-sfr and PNOS-neo which aremarkers DNAs with respectively PSSU and PNOS as second promoters; andPTA29-Barnase gene which is a male-sterility DNA under the control ofPTA29 as a first promoter. Expression in tapetum cells of themale-sterility DNA under the control of the TA29 promoter will produceBarnase selectively in the tapetum cells so that Barnase will interferewith the metabolism of these cells.

EXAMPLE 13

Introduction of the Chimaeric DNA Sequence of Example 12 into Tobaccoand Oilseed Rape

As described in Example 11, a recombinant Agrobacterium strain wasconstructed by mobilizing pTTM8 (from Example 12) from E. coli intoAgrobacterium C58C1 Rif^(R) containing pMP90 (Koncz and Schell (1986)Mol. Gen. Genetics 204, 383-396). The resulting strain (named “A3135”),harboring pMP90 and PTTM8, is used for tobacco leaf disc transformationand for oilseed rape transformation. Transformed calli and shoots areselected using 5 mg/l phosphinothricin and 100 ug/ml kanamycin. That theBarnase gene is not expressed in the transformed herbicide-resistantcalli and shoots is shown by their growth.

The transformed shoots are rooted, transferred to soil in the greenhouseand grown until they flower. The flowers of both the tobacco and oilseedrape are examined, and a phenotype is observed for the transformedplants that is essentially the same as the phenotype of the transformedtobacco plants described in Example 10. This shows that the TA29promoter is capable of directing expression of the heterologous Barnasegene selectively in tapetum cells of the plants, thereby rendering theplants male-sterile.

EXAMPLE 14

Construction of a Chimaeric DNA Sequence of pTA29 and a Gene EncodingPapain

A plasmid named “pTVEP1”, shown in FIG. 9A, is constructed by assemblingthe following well known fragments with PTA29:

1. a vector fragment, including T-DNA border sequences derived frompGSC1700 and in which the β-lactamase gene (1′ of FIG. 9A) has beeninactivated by insertion of a DNA sequence into its ScaI site;

2. the chimaeric sequence (no. 3) of Example 5, containing the PSSUpromoter, the herbicide resistance gene sfr and the 3′ end of T-DNA gene7.

3. the chimaeric sequence (no. 4) of Example 5, containing the PNOSpromoter, the neo gene and the 3′ end of the octopine synthase gene; and

4. a chimaeric sequence, containing the PTA29 promoter cassette fromExample 3, fused in frame with:

a) a papain gene from Carica papaya fruit, encoding the papain zymogenwhich is a plant endopeptidase (Cohen et al (1986) Gene 48, 219-227)capable of attacking peptide, as well as ester, bonds; the followingmodifications are made in the DNA sequence of Cohen et al (1986) usingsite directed mutagenesis as described in Example 3:

i. the nucleotide A, position-1 upstream of the first ATG codon, ismutated into nucleotide C in order to obtain a suitable NcoI cloningsite; and

ii. the GAA codons encoding glutamate at positions 47, 118, 135,respectively, are mutated into CAA codons encoding glutamine; and

b) the 3′ end of the nopaline synthase gene of Example 9.

pTVEP1 is a binary type T-DNA vector containing, within the T-DNA bordersequences, three chimaeric sequences: PSSU-sfr and PNOS-neo which aremarker DNAs encoding dominant selectable markers for planttransformations, under the control of respectively PSSU and PNOS assecond promoters; and PTA29-Papain gene which is a male-sterility DNAunder the control of PTA29 as a first promoter. Expression in tapetumcells of the male-sterility DNA under the control of the TA29 promoterwill produce an endopeptidase (the papain zymogen) that will cleaveproteins in the tapetum cells, thus leading to the death of these cells.

A plasmid named “pTVEP2”, shown in FIG. 9B, is also constructed byassembling the following well known fragments with PTA29:

1. a vector fragment, including T-DNA border sequences derived frompGSC1700 and in which the β-lactamase gene (1′ of FIG. 9B) has beeninactivated by insertion of a DNA sequence into the ScaI site;

2. the chimaeric sequence (no. 3) of Example 5, containing the PSSUpromoter, the herbicide resistance gene sfr and the 3′ end of T-DNA gene7;

3. the chimaeric sequence (no. 4) of Example 5, containing the PNOSpromoter, the neo gene, and the 3′ end of the octopine synthase gene;and

4. a chimaeric sequence, containing the PTA29 promoter cassette ofExample 3, fused in frame with:

a) a papain gene from Carica papaya fruit, encoding the active proteinof the papain zymogen; the following modifications are made in the DNAsequence of Cohen et al (1986), using site directed mutagenesis asdescribed in Example 3:

i. the AAT codon encoding Asn, upstream of the first Ile residue of theactive protein, is mutated into a GAT codon, which provides a suitableEcoRV cloning site (GAT ATC). The EcoRV engineered site is fuseddirectly to the pTA29 cassette in order to obtain a direct in framefusion of the promoter with the sequence encoding the active protein ofthe papain zymogen; and

ii. the GAA codons encoding glutamate at positions 47, 118, 135respectively, are mutated into CAA codons encoding glutamine: and

b) the 3′ end of the nopaline.synthase gene of Example 9.

pTVEP2, like pTVEP1, is a binary type T-DNA vector containing, withinthe T-DNA border sequences, three chimaeric genes: PSSU-sfr and PNOS-neoencoding dominant selectable markers for plant transformations; andPTA29-Papain gene which encodes an endopeptidase that will cleaveproteins in the tapetum cells, thus leading to the death of these cells.

EXAMPLE 15

Introduction of the Chimaeric DNA Sequences of Example 14 into Tobaccoand Oilseed Rape

As described in Example 11, pTVEP1 and pTVEP2, are each mobilized fromE. coli into separate Agrobacterium C58C1 Rif^(R) carrying pMP90.

The resulting strains, harboring pMP90 with pTVEP1 and pMP90 withpTVEP2, are used to transform tobacco and oilseed rape following theprocedures of Examples 11 and 13. That the papain genes are notexpressed in transformed herbicide- and kanamycin-resistant calli,shoots and roots is shown by their growth.

The transformed plants are transferred into the greenhouse and grown insoil until they flower. The flowers of both the tobacco and oilseed rapeare examined, and phenotypes are observed for the transformed plantsthat are essentially the same as the phenotype of the transformedtobacco plants described in Example 10. This shows that the TA29promoter is capable of directing expression of the heterologous papaingenes in pTVEP1 and pTVEP2 selectively in tapetum cells of the plants,thereby rendering the plants male-sterile.

EXAMPLE 16

Construction of a Chimaeric DNA Sequence of pTA29 and a Gene EncodingEcoRI

A plasmid named “pTVE63”, shown in FIG. 10A, was constructed byassembling the following well known fragments with PTA29:

1. a vector fragment, including T-DNA border sequences derived frompGSC1701A2 (European patent application 87/115985.1);

2. the chimaeric sequence (no. 3) of Example 5, containing the PSSUpromoter, the herbicide-resistance gene sfr and the 3′ end of T-DNA gene7;

3. the chimaeric sequence (no. 4) of Example 5, containing the PNOSpromoter, the neo gene and the 3′ end of the octopine synthase gene;

4. a chimaeric sequence, containing the pTA29 promoter cassette ofExample 3, fused in frame with:

a) a gene encoding the EcoRI restriction endonuclease from an E. coli(Green et al (1981) J. Biol. Chem. 256, 2143-2153; Botterman and Zabeau(1985) Gene 37, 229-239) and capable of recognizing and cleaving thetarget sequence GAATTC on a double stranded DNA; the followingmodifications were made in the DNA sequence of Green et al (1981) usingsite directed mutagenesis as described in Example 3:

i. the nucleotides of the ATG initiation codon were replaced by ATGCA,creating a NsiI site at the initiation codon and yielding the followingnucleotide sequences:

ATCCA,TCT,AAT . . . ; and

ii. the HindII-HindIII fragment of the EcoRI gene cloned in pEcoR12(Botterman and Zabeau, 1985) was cloned into the pMAC5-8 site directedmutagenesis vector; and

b) the 3′ end of the nopaline synthase gene of Example 9; and

5. a gene encoding an EcoRI methylase under the control of its naturalpromoter (Botterman and Zabeau (1985) Gene 37, 229-239) which is capableof inhibiting the activity of EcoRI in E. coli or Agrobacterium, inorder to overcome potential leaky expression of the EcoRI gene inMicroorganisms.

pTVE63 is a binary type T-DNA vector containing, within the T-DNA bordersequences, three chimaeric sequences: PSSU-sfr and PNOS-neo which aremarker DNAs under the control of respectively PSSU and PNOS as secondpromoters; and PTA29-EcoRI gene which is a male-sterility DNA under thecontrol of PTA29 as a first promoter. Expression of the male-sterilityDNA under the control of the TA29 promoter in tapetum cells will producethe EcoRI restriction endonuclease which will cleave double stranded DNAat the GAATTC sites (see for review of type II restriction Modificationsystems: Wilson (1988) TIG 4 (11), 314-318) of the tapetus cells, thusleading to the death of these cells.

A plasmid named pTVE62, shown in FIG. 10B, was also constructed byassembling the following well known fragments with PTA29:

1. a vector fragment, including T-DNA border sequences derived frompGSC1701A2;

2. the chimaeric sequence (no. 3) of Example 5, containing the PSSUpromoter, the herbicide-resistance gene sfr and the 3′ end of T-DNA gene7;

3. the chimaeric sequence (no. 4) of Example 5, containing the PNOSpromoter, the neo gene and the neo 3′ end of the octopine synthase gene;

4. a chimaeric sequence, containing the pTA29 promoter cassette ofExample 3, fused in frame with a gene fragment encoding the transitpeptide of the Mn-superoxide dismutase (“Mn-SOD”) which is a NcoI-PstIfragment of a HpaI-HindIII fragment from pSOD1 (Bowler et al (1989) EmboJ. 8, 31-38); the following modifications were made in the DNA sequenceof Bowler et al using site directed mutagenesis as described in Example3:

i. the AA nucleotides located upstream at position −2 and −1 of the ATGinitiation codon were changed to CC nucleotides creating a NcoI site atthe initiation codon and yielding the following nucleotide sequences:

-CCATGGCACTAC      NcoI

ii. the T,TCG,CTC, nucleotides located immediately downstream of theprocessing site of the transit peptide were changed to C,TGC,AGC,creating a PstI site behind the processing site and yielding the thefollowing nucleotide sequences:

in which the arrow indicates the processing site of the transit peptidesequence and the upper line the aminoacid sequence corresponding withthe Mn-SOD coding sequence; the NcoI-PstI fragment was also fused inframe with a gene encoding the EcoRI restriction endonuclease from E.coli (Greene et al (1981) J. Biol. Chem. 256, 2143-2153; Botterman andZabeau (1985) Gene 37, 229-239) and capable of recognition and cleavageof the target sequence GAATTC on a double stranded DNA, as found inpTVE63; and

b) the 3′ end of the nopaline synthase gene of Example 9; and

5. a gene encoding the EcoRI methylase under the control of its naturalpromoter (Botterman and Zabeau, 1985) which is capable of inhibiting theactivity of EcoRI in E. coli or Agrobacterium, in order to overcomepotential leaky expression of the EcoRI gene in microorganisms, thisgene being inserted into the vector fragment outside the bordersequences.

pTVE62 is a binary type T-DNA vector containing, within the bordersequences, three chimeric sequences: PSSU-sfr and PNOS-NPTII which aremarker DNAs under the control of respectively PSSU and PNOS as secondpromoters; and. pTA29-transit peptide-EcoRI endonuclease gene which is amale-sterility DNA having PTA29 as a first promoter and a transitpeptide-encoding sequence between then. Expression of the male-sterilityDNA under the control of the TA29 promoter in tapetum cells will producea restriction endonuclease which will be targeted into the mitochondriaof the tapetum cells and cleave the double stranded DNA at the GAATTCsites in such cells. This will lead to the death of these cells.

EXAMPLE 17

Introduction of the Chimaeric DNA Sequences of Example 16 into Tobaccoand Oilseed Rape

As described in Examples 11 and 15, pTVE62 and pTVE63, were mobilizedfrom E. coli into Agrobacterium C58C1 Rif^(R) carrying pMP90. Theresulting strains, harboring pTVE62 with pMP90 and pTVE62 (with pMP90,were used to transform tobacco and are used to transform oilseed rapefollowing the procedures described in Examples 11 and 13. That the EcoRIendonuclease genes were not expressed in transformed herbicide- andkanamycin-resistant calli, shoots and roots is shown by their growth.

The transformed plants are transferred into the greenhouse and grown insoil until they flower. The flowers of both the tobacco and oilseed rapeare examined, and phenotypes are observed for the transformed plantsthat are essentially the same as of the transformed tobacco plantsdescribed in Example 10. This shows that the TA29 promoter is capable ofdirecting expression of the heterologous EcoRI endonuclease geneselectively in the tapetum cells of the plants transformed with pTVE62and pTVE63, thereby rendering the plants male-sterile.

Needless to say, this invention is not limited to the transformation ofany specific plant(s). The invention relates to any plant, the nucleargenome of which can be transformed with a male-sterility DNA under thecontrol of a first promoter that can direct expression of themale-sterility DNA selectively in the plant's stamen cells, whereby theplant can be both self-pollinated and cross-pollinated. For example,this invention relates to plants such as potato, tomato, oilseed rape,alfalfa, sunflower, cotton, celery, onion, corn, soybean, tobacco,brassica vegetables and sugarbeet.

Also, this invention is not limited to the specific plasmids and vectorsdescribed in the foregoing Examples, but rather encompasses any plasmidsand vectors containing the male-sterility DNA under the control of thefirst promoter.

Furthermore, this invention is not limited to the specific promotersdescribed in the foregoing Examples, such as the TA29 promoter, butrather encompasses any DNA sequence encoding a promoter capable ofdirecting expression of the male-sterility DNA selectively in stamencells. In this regard, this invention encompasses the DNA sequence ofthe TA29 promoter of FIG. 3A, as well as any equivalent DNA sequences,such as that of the TA13 promoter of FIG. 3B and the TA 26 promoter ofFIG. 3C, which can be used to control the expression of themale-sterility DNA selectively in tapetum cells of a plant. Indeed, itis believed that the DNA sequences of the TA29, TA26 and TA13 promoterscan be modified by: 1) replacing some codons with others that codeeither for the same amino acids or for other amino acids; and/or 2)deleting or adding some codons; provided that such modifications do notsubstantially alter the properties of the encoded promoter forcontrolling tapetum-specific expression of a male-sterility.

In addition, this invention is not limited to the specificmale-sterility DNAs described in the foregoing Examples but ratherencompasses any DNA sequence encoding a first RNA, protein orpolypeptide which disturbs significantly the metabolism functioningand/or development of a stamen cell in which it is produced, under thecontrol of the first promoter.

Also, this invention is not limited to the specific marker DNAsdescribed in the foregoing Examples but rather encompasses any dnasequence encoding a second RNA, protein or polypeptide which confers onat least a specific plant tissue or specific plant cells, in which suchDNA sequence is expressed, a distinctive trait compared to such aspecific plant tissue or specific plant cells in which such DNA sequenceis not expressed.

What is claimed is:
 1. A cell of a plant, the nuclear genome of saidcell being transformed with a foreign DNA sequence; said foreign DNAsequence comprising: a) a male-sterility DNA encoding a first protein orpolypeptide which, when produced or overproduced in a stamen cell ofsaid plant, disturbs significantly one or more activities of said stamencell selected from the group consisting of the metabolism, functioningand development of said stamen cell; and b) a first promoter capable ofdirecting expression of said male-sterility DNA selectively in stamencells of said plant; said male-sterility DNA being in the sametranscriptional unit as, and under the control of, said first promoter;whereby said stamen cells of said plant are killed or disabled byexpression of said male sterility DNA selectively in said stamen cellsof said plant, so as to render said plant incapable of producing fertilemale gametes.
 2. The cell of claim 1, wherein said foreign DNA sequenceis a foreign chimaeric DNA sequence.
 3. The cell of claim 2, whereinsaid first promoter is capable of directing expression of saidmale-sterility DNA in one or more types of stamen cells of said plantselected from the group consisting of anther, pollen and filament cellsof said plant.
 4. The cell of claim 3, wherein said first promoter iscapable of directing expression of said male-sterility DNA in one ormore types of stamen cells of said plant selected from the groupconsisting of tapetum and anther epidermal cells of said plant.
 5. Thecell of claim 1, wherein said foreign DNA sequence also comprises: c) amarker DNA encoding a marker RNA, protein or polypeptide which, whenpresent in a specific tissue or in specific cells of said plant, renderssaid plant easily separable from other plants which do not contain saidmarker RNA, protein or polypeptide in said specific tissue or specificcells; and d) a second promoter capable of directing expression of saidmarker DNA in said specific tissue or specific cells; said marker DNAbeing in the same transcriptional unit as, and under the control of,said second promoter.
 6. The cell of claim 5, wherein said marker DNAand said second promoter are in the same genetic locus as saidmale-sterility DNA and said first promoter.
 7. The cell of claim 6,wherein said foreign DNA sequence further comprises additional DNAselected from the group consisting of: e) a first DNA encoding a transitpeptide capable of transporting said first protein or polypeptide into achloroplast or mitochondria of said stamen cell; said first DNA being inthe same transcriptional unit as said male-sterility DNA and said firstpromoter; said first DNA also being between said male-sterility DNA andsaid first promoter; and f) a second DNA encoding a transit peptidecapable of transporting said marker protein or polypeptide into achloroplast or mitochondria of said specific tissue or specific cells;said second DNA being in the same transcriptional unit as said markerDNA and said second promoter; said second DNA being between said markerDNA and said second promoter.
 8. The cell of claim 1, wherein saidmale-sterility DNA encodes a compound selected from the group consistingof: an RNase, a DNase, a protease, a glucanase, a lipase, a lipidperoxidase, a cell wall inhibitor, and a bacterial toxin.
 9. The cell ofclaim 1, wherein said male-sterility DNA encodes an enzyme whichcatalyses synthesis of a phytohormone.
 10. The cell of claim 9, whereinsaid male-sterility DNA encodes an enzyme encoded by gene 1, gene 2 orgene 4 of Agrobacterium T-DNA.
 11. The cell of claim 5, wherein saidmarker DNA is selected from the group consisting of: an herbicideresistance gene; a gene encoding a modified target enzyme for anherbicide having a lower affinity for the herbicide than does thecorresponding unmodified target enzyme; a gene encoding a protein orpolypeptide conferring a color to said specific tissue or specificcells; a gene encoding a protein or polypeptide conferring a stresstolerance to said plant; and a gene encoding a protein or polypeptideconferring a disease resistance or pest resistance to said plant. 12.The cell of claim 11, wherein said marker DNA is selected from the groupconsisting of: an sfr gene; an sfrv gene; a gene encoding a modified5-enolpyruvylshikmate-3 phosphate synthase as a target enzyme forglyphosate; a gene encoding a modified glutamine synthetase as a targetenzyme for a glutamine synthetase inhibitor; the A1 gene; the GUS gene;a gene encoding Mn-superoxide dismutase; a gene encoding a Bacillusthuringiensis endotoxin that confers insect resistance; and a geneencoding a bacterial peptide that confers bacterial resistance.
 13. Thecell of claim 1, wherein said first promoter is selected from the groupconsisting of PTA29, PTA26, and PTA13.
 14. The cell of claim 5, whereinsaid second promoter is selected from the group consisting of: aconstitutive promoter, a wound-inducible promoter, a promoter whichdirects gene expression selectively in plant tissue havingphotosynthetic activity, and a promoter which directs gene expressionselectively in leaf cells, petal cells or seed cells.
 15. The cell ofclaim 14, wherein said second promoter is selected from the groupconsisting of: a CaMV 35S promoter, a CaMV 35S'3 promoter, PNOS, POCS, aTR1′ promoter, a TR2′ promoter, a ribulose bisphosphate carboxylase SSUpromoter, and a promoter which directs expression selectively in seedcoat cells.
 16. The cell of claim 1, which comes from a cultureconsisting essentially of said cells; each of said cells from saidculture being transformed with said foreign DNA sequence and beingcapable of being used to regenerate said plant as a homozygous plant.17. The cell of claim 16, which comes from an anther cell culture. 18.The cell of claim 16, wherein said first promoter is capable ofdirecting expression of said male-sterility DNA selectively in pollencells of said plant after meiosis of said pollen cells.
 19. A vectorsuitable for transforming a cell of a plant; said vector comprising saidforeign DNA sequence of claim
 1. 20. The vector of claim 19 selectedfrom the group consisting of pTTM4, pTTM6, pTTM6A⁻, pTTM8, pTVEP1,pTVEP2, pTVE62 and pTVE63.
 21. A plant cell culture, consistingessentially of the cells of claim
 1. 22. A plant consisting essentiallyof cells, the nuclear genome of said cells being transformed with aforeign DNA sequence; said foreign DNA sequence comprising: a) amale-sterility DNA encoding a first protein or polypeptide which, whenproduced or overproduced in a stamen cell of said plant, disturbssignificantly one or more activities of said stamen cell selected fromthe group consisting of the metabolism, functioning and development ofsaid stamen cells; and b) a first promoter capable of directingexpression of said male-sterility DNA selectively in stamen cells ofsaid plant; said male-sterility DNA being in the same transcriptionalunit as, and under the control of, said first promoter; whereby saidstamen cells of said plant are killed or disabled by expression of saidmale-sterility DNA selectively in said stamen cells of said plant; so asto render said plant incapable of producing fertile male gametes.
 23. Aseed of the plant of claim
 22. 24. A seed of claim 23 which is a hybridseed.
 25. A hybrid plant obtained by growing the hybrid seed of claim24.
 26. The cell of claim 1, wherein said male-sterility DNA is: a) notnaturally found under the control of said first promoter; or b) notnaturally found in the same genetic locus as said marker DNA; or c) notnaturally found under the control of said first promoter and in the samegenetic locus as said marker DNA.
 27. The cell of claim 1, wherein saidmale-sterility DNA encodes said first protein or polypeptide whichinterferes significantly with the metabolism or the functioning or boththe functioning and the metabolism of cells in which it is present; andsaid first promoter is capable of directing expression of saidmale-sterility DNA selectively in anther cells of said plant.
 28. Thecell of claim 13 wherein said first promoter is PTA29.
 29. The cell ofclaim 8, wherein said male-sterility DNA encodes a compound selectedfrom the group consisting of: RNase T1; Barnase; an endonuclease; apapain; and phospholipase A2.
 30. The cell of claim 29, wherein saidmale-sterility DNA encodes EcoRI, papain Zymogen or papain activeprotein.
 31. The plant of claim 22, wherein said foreign DNA sequence isa foreign chimeric DNA sequence.
 32. The plant of claim 31, wherein saidfirst promoter is capable of directing expression of said male-sterilityDNA in one or more types of stamen cells of said plant selected from thegroup consisting of anther, pollen and filament cells of said plant. 33.The plant of claim 31, wherein said first promoter is capable ofdirecting expression of said male-sterility DNA in one or more types ofstamen cells of said plant selected from the group consisting of tapetumand anther epidermal cells of said plant.
 34. The plant of claim 22,wherein said foreign DNA sequence also comprises: c) a marker DNAencoding a marker RNA, protein or polypeptide which, when present in aspecific tissue or in specific cells of said plant, renders said planteasily separable from other plants which do not contain said marker RNA,protein or polypeptide in said specific tissue or specific cells; and d)a second promoter capable of directing expression of said marker DNA insaid specific tissue or specific cells; said marker DNA being in thesame transcriptional unit as, and under the control of, said secondpromoter.
 35. The plant of claim 34, wherein said marker DNA and saidsecond promoter are in the same genetic locus as said male-sterility DNAand said first promoter.
 36. The plant of claim 35, wherein said foreignDNA sequence further comprises additional DNA selected from the groupconsisting of: e) a first DNA encoding a transit peptide capable oftransporting said first protein or polypeptide into a chloroplast ormitochondria of said stamen cell; said first DNA being in the sametranscriptional unit as said male-sterility DNA and said first promoter;said first DNA also being between said male-sterility DNA and said firstpromoter; and f) a second DNA encoding a transit peptide capable oftransporting said marker protein or polypeptide into a chloroplast ormitochondria of said specific tissue or specific cells; said second DNAbeing in the same transcriptional unit as said marker DNA and saidsecond promoter; said second DNA being between said marker DNA and saidsecond promoter.
 37. The plant of claim 22, wherein said male-sterilityDNA encodes a compound selected from the group consisting of: an RNase,a DNase, a protease, a glucanase, a lipase, a lipid peroxidase, a cellwall inhibitor and a bacterial toxin.
 38. The plant of claim 22, whereinsaid male-sterility DNA encodes an enzyme which catalyses synthesis of aphytohormone.
 39. The plant of claim 38, wherein said male-sterility DNAencodes an enzyme encoded by gene 1, gene 2 or gene 4 of AgrobacteriumT-DNA.
 40. The plant of claim 34, wherein said marker DNA is selectedfrom the group consisting of: an herbicide resistance gene; a geneencoding a modified target enzyme for an herbicide having lower affinityfor the herbicide than does the corresponding unmodified target enzyme;a gene encoding a protein or polypeptide conferring a color to saidspecific tissue or specific cells; a gene encoding a protein orpolypeptide conferring a stress tolerance to said plant; and a geneencoding a protein or polypeptide conferring a disease resistance orpest resistance to said plant.
 41. The plant of claim 34, wherein saidmarker DNA is selected from the group consisting of: an sfr gene; ansfrv gene; a gene encoding a modified 5-enopyruvylshikimate-3 phosphatesynthase as a target enzyme for glyphosate; a gene encoding a modifiedglutamine synthetase as a target for a glutamine synthetase inhibitor;the A1 gene; the GUS gene; a gene encoding Mn-superoxide dismutase; agene encoding a Bacillus thuringiensis endotoxin that confers insectresistance; and a gene encoding a bactericidal peptide that confersbacterial resistance.
 42. The cell of claim 5, wherein said marker DNAis a gene encoding resistance to glutamine synthetase inhibitors. 43.The plant of claim 34, wherein said marker DNA is a gene encodingresistance to glutamine synthetase inhibitors.
 44. The plant of claim22, wherein said first promoter is selected from the group consisting ofPTA29, PTA26 and PTA13.
 45. The plant of the claim 34, wherein saidsecond promoter is selected from the group consisting of: a constitutivepromoter, a wound-inducible promoter, a promoter which directs geneexpression selectively in plant tissue having photosynthetic activity,and a promoter which directs gene expression selectively in leaf cells,petal cells or seed cells.
 46. The plant of claim 45, wherein saidsecond promoter is selected from the group consisting of: a CaMV 35Spromoter, a CaMV 35S'3 promoter, PNOS, POCS, a TR1′ promoter, a TR2′promoter, a ribulose bisphosphate carboxylase SSU promtoer, and apromoter which directs expression selectively in seed coat cells. 47.The plant of claim 22, wherein said male-sterility DNA encodes saidfirst protein or polypeptide which interferes significantly with themetabolism or the functioning or both the functioning and the metabolismof cells in which it is present; and said first promoter is capable ofdirecting expression of said male-sterility DNA selectively in anthercells of said plant.
 48. The plant of claim 44 wherein said firstpromoter is PTA29.
 49. The plant of claim 37 wherein said male-sterilityDNA encodes a compound selected from the group consisting of: RNase T1;Barnase; an endonuclease; a papain; and phospholipase A2.
 50. A plantconsisting essentially of cells, the nuclear genome of said cells beingtransformed with a foreign DNA sequence; said foreign DNA sequencecomprising: a) a male-sterility DNA encoding barnase; b) a firstpromoter capable of directing expression of said male-sterility DNAselectively in stamen cells of said plant; said male-sterility DNA beingin the same transcriptional unit as, and under the control of, saidfirst promoter; whereby said stamen cells of said plant are killed ordisabled by expression of said male-sterility DNA selectively in saidstamen cells of said plant, so as to render said plant incapable ofproducing fertile male gametes.
 51. The plant of claim 50, wherein saidfirst promoter is a tapetum-specific promoter.
 52. The plant of claim50, wherein said foreign DNA sequence further comprises a marker DNAconsisting of an herbicide resistance gene.
 53. The plant of claim 52,wherein said marker DNA is an sfr gene or an sfrv gene.
 54. The plant ofclaim 50, wherein said first promoter is PTA29, PTA26 or PTA13.
 55. Theplant of claim 22, wherein said first promoter is a tapetum-specificpromoter.
 56. The plant of claim 22, wherein said male-sterility DNAencodes a protein which is cytotoxic to plant cells.
 57. The plant ofclaim 22, wherein said male-sterility DNA encodes a ribonuclease. 58.The plant of claim 34, wherein said marker DNA is a herbicide resistancegene.
 59. The plant of claim 34, wherein said marker DNA is a geneconferring resistance to a glutamine synthetase inhibitor.
 60. The plantof claim 59, wherein said marker DNA is a gene conferring resistance tophosphinothricine.
 61. The plant of claim 58, wherein said marker DNA isan sfr gene or an sfrv gene.
 62. The seed of a plant of any one ofclaims 31-61.
 63. The seed of a plant of any one of claims 31-61, whichis a hybrid seed.
 64. The plant grown from the seed of claim 62, whichis a hybrid plant.
 65. The cell of claim 11, wherein said marker DNA isa gene conferring resistance to a glutamine synthetase inhibitor. 66.The cell of claim 65, wherein said marker DNA is a gene conferringresistance to phosphinothricine.
 67. The plant grown from the seed ofclaim 63, which is a hybrid plant.